The body of this research program has focused on the critical relationship between lipids and cellular proteostasis of the presynaptic protein alpha-synuclein (alpha-syn) and how it relates to PD. A fundamental question that is addressed is the role of membranes in promoting misfolding and aggregation of alpha-syn. We have carried out detailed investigations of membrane interactions and amyloid formation of alpha-syn that have provided residue-specific information and molecular insights into the mechanism of aggregation. Due to the complexity of the amyloid problem, the tools with which we attack have included molecular biology, steady-state and time-resolved fluorescence spectroscopy, nuclear magnetic resonance spectroscopy, electron microscopy, neutron reflectometry (NR), and mass spectrometry. We are moving towards translating sophisticated spectroscopic measurements into a cellular context as well as designing biochemical analyses of whole tissue lysates. In illuminating the relationship between lipids and alpha-syn and how it relates to PD, we are developing a chemical understanding in how specific phospholipids modulate protein structure, membrane binding, and aggregation propensity through a number of different studies summarized below. 1. We are continuing our efforts in pushing the utility of NR, a scattering method, to investigate membrane interactions of alpha-syn. NR is unique among available structural techniques in that it allows the characterization of membrane-associated proteins bound to actual lipid bilayers in an aqueous environment, as opposed to proteins in solid state or detergent solubilized forms currently prevalent in the field. The reflection of neutrons from bilayer membranes simultaneously probes the protein and the bilayer and readily distinguishes the layers of lipid acyl chains and phospholipid headgroups as well as the membrane bound proteins. Both the extent of polypeptide insertion into the bilayer and extension into the aqueous surrounding can be measured. However, the specific polypeptide regions involved in membrane association are not distinguishable and that information must be inferred from other experimental methods. To address this, we are exploiting the high sensitivity of neutron scattering length density differences for deuterons vs. protons by producing partial or segmental deuterated alpha-syn via native chemical ligation. Using two segmentally deuterated proteins, identification of alpha-syn regions associated with the lipid bilayer have been accomplished. 2. We are studying the ability of alpha-syn to drive membrane curvature. Different lipid structures such as bilayer discs (nanodiscs), micellar tubes (7 nm), and large tubules (20 nm) have been observed for membrane remodeling induced by alpha-syn in vitro. We are interested in the interplay between protein structure and membrane deformation and how this process is regulated by phospholipid composition. Different lipid compositions including those that stabilize alpha-syn helical formation are being examined such as phosphatidic acid, and phosphatidylglycerol. To further delineate the membrane remodeling mechanism induced by alpha-syn, the effects of lipid physical properties (e.g. bilayer fluidity and phase state) are also investigated. 3. There may be an intimate relationship between membrane curvature generation and membrane curvature sensing by alpha-syn. That is, does alpha-syn induce membrane curvature because it prefers curved surfaces? One possibility is that smaller vesicles have more defects in their surfaces which facilitate alpha-syn insertion and folding. Or, could membrane curvature sensing be coordinated by specific lipids? We aim to discern the biophysical basis for curvature sensing by alpha-syn. Towards this goal, we have developed a method to track freely-diffusing vesicles down to a radius of 10 nm through an easily constructed confinement chamber. Vesicle size is directly measured through particle tracking of the fluorescent vesicles (i.e. diffusion coefficient) rather than by fluorescence intensity calibration. Neither surface attachment of vesicles nor preparation/separation of varying sizes of vesicles is needed. Heterogeneous binding behaviors averaged out in the bulk can be revealed on the single-vesicle level. Feasibility of this method has been demonstrated on counting and sizing of high-density, low-density, and very low-density lipoproteins. We will now extend this method to the simultaneous measurements of alpha-syn binding to two different vesicles compositions (i.e. competition experiment) and investigate whether the preference for smaller radius is coupled to lipid specificity. A three-color system composed of two types of synthetic vesicles incorporated with two lipophilic fluorophores, DiI and DiD, and alpha-syn covalently modified at Cys136 with Alexa488 has been implemented. Since small particles are prolific in human physiology, we envisage this method will have broad applicability as free particle tracking is especially advantageous when working with native particles. 4. We have completed the construction of flexible instrumentation that couples a Raman spectrometer to an epifluorescence microscope with a nanopositioning piezo stage. This setup allows us to measure Raman vibrational spectroscopic data at several excitation wavelengths and collect spatial information simultaneously. This powerful approach enables protein secondary structural changes to be monitored, allowing us to distinguish whether the protein has adopted a beta-sheet rich form indicative of amyloid structure, while pinpointing its intracellular spatial location. This direct spectroscopic characterization will yield chemical (i.e. specific stretching frequencies) and structural information which fluorescence imaging cannot provide on its own.